ABSTRACT ? PROJECT 3 The goal of this project is to develop efficient and safe gene therapy strategies for the treatment of nonmalignant hematopoietic diseases. We hypothesize that different nonmalignant hematopoietic diseases will require varied approaches to gene therapy to achieve maximum therapeutic benefit while maintaining safety. We focus our studies on the dog because it allows for a direct translation to the clinic and because several canine disease models are available that bear a close resemblance to the human disease. Over the past 15 years we have demonstrated efficient ex vivo gene transfer into hematopoietic stem/progenitor cells (HSCs) and long-term in vivo repopulation with all 3 commonly used integrating retrovirus systems: gammaretrovirus (RV), lentivirus (LV), and foamy virus (FV) vectors. Our retrovirus integration site (RIS) studies in the dog model have shown that LV and FV vectors have a favorable integration site pattern compared to RV vectors, which is critical given the development of leukemia after RV-based HSC gene therapy in patients with X-linked severe combined immunodeficiency (SCID-X1). We will focus on LV vectors because of their advanced design, clinical utility, and the availability of novel pseudotypes. One focus of this proposal is the development and testing of novel conditioning regimens. For most genetic diseases, myelosuppression to reduce endogenous HSCs will be required for successful engraftment of ex vivo modified HSCs. Here we will study novel conditioning regimens based on treosulfan and alpha emitter astatine-211, used in Projects 1 and 2 in the allogeneic transplant setting. Also, given that the number of HSCs is limited for some diseases like Fanconi anemia, we will also pursue ex vivo expansion of HSCs and study whether ex vivo HSC expansion can enhance engraftment and further reduce the level of pretransplant conditioning required for engraftment. As an alternative approach especially for SCID-X1 we will also evaluate direct in vivo administration of lentivirus vectors. We have two canine disease models available in which to study our improved expansion and conditioning strategies, the pyruvate kinase (PK) deficiency model, a red blood cell disorder which has some of the same issues as hemoglobinopathies, and the SCID-X1 model which very closely resembles patients with SCID-X1.